The present invention relates generally to measuring temperature dependent properties of liquids or solid samples contained within a liquid bath or otherwise thermally coupled to the holding fixture. More particularly, the apparatus and methods of the invention describe an apparatus including a liquid containing fixture with a predetermined temperature gradient thereacross equipped with an optical means to measure temperature-dependent optical properties of the liquid of interest contained therein.
Calorimetry is a well-known method of evaluating the thermal and thermodynamic properties of liquids. Prior art in calorimetry is well described in numerous textbooks on physics and physical chemistry and in monographs devoted to the subject. When the liquids display favorable optical properties, methods for indirect determination of thermal and thermodynamic properties are known. Several formulations based on the van't Hoff equation for example have been described in the art aiming at extracting thermodynamic information for non-calorimetric observables measured as a function of temperature. The words “observable” and “property” are used interchangeably in this description and have the same meaning. These van't Hoff methods can be advantageously applied to temperature-dependent spectroscopic data.
Liquids present a variety of temperature dependent optical properties. Several nonlimiting examples are discussed herewith. The refractive index of most pure liquids, mixtures and solutions depends on temperature. Further, liquids containing one or more optically active components display temperature dependent birefringence. Liquids comprising or containing chromophores or fluorophores display temperature dependent absorbance or fluorescence properties. The molecular origins of the temperature dependent changes in absorbance or fluorescence spectra of a particular liquid may arise from one or more of several processes. Examples of thermochromic reactions include ligand substitution reactions such as observed when hexaaquacobalt (II) is heated in mixtures of water and primary alcohols. Other examples include temperature dependent changes in ionization state of chromophores or fluorophors, which are coupled to optical changes. Other examples include processes in which molecular complexes change conformation as a function of temperature resulting in changes in optical properties. Such processes frequently involve changes in the solvent exposure of chromophores or fluorophores attached to polymers. Examples include the temperature dependent conformational changes in proteins and nucleic acids which alter the chemical environment of intrinsic (such as amino acid side chains or covalently bound cofactors in proteins or nucleobases in nucleic acids) or extrinsic (such as noncovalently bound cofactors, or drug molecules) chromophores or fluorophores. Macromolecule conformations may be mediated by small molecule effectors as a function of temperature. Such small molecule mediated effects on optical properties are observed frequently with proteins and nucleic acids but may also be observed in synthetic polymers or carbohydrates an example of which is the changes observed when iodine-starch mixtures are subjected to temperature changes. In the above examples, if the chromophore or fluorophore is optically active or bound to an optically active substrate temperature dependent dichroism or anisotropy may be observable.
Current temperature-dependent spectroscopic and calorimetric methods are laborious and material intensive. In most cases, spectroscopic methods require measuring an optical property of a liquid at one particular temperature preset point and then repeating this measurement for another temperature point until the entire characteristic of the optical property is obtained. Complex sample holding cells are described in the prior art allowing maintaining the temperature of the sample liquid at a desired level. An example of such a cell is described in the U.S. Pat. No. 5,192,910 and includes a sophisticated system for maintaining the same temperature throughout the entire liquid volume. A significant amount of time is needed to achieve and stabilize the temperature of the next measurement point and therefore the entire characteristic can not be obtained quickly. Improvements in sample throughput are therefore needed to make high-throughput thermodynamics practical. A throughput increase of at least two orders of magnitude is required. A need exists in a liquid spectroscopy technology permitting a significant acceleration in optical and thermodynamic characterization of liquids.
Another disadvantage of the temperature dependent optical methods of the prior art is in the discrete nature of measurements. Only certain temperature points are available on the curve and therefore in transitional phases it is quite difficult to obtain information about the property of interest with sufficient resolution without either prior knowledge of the point of transition and its breadth or the time consuming collection of high resolution data outside the range of interest. This also pertains to so-called “zooming”, when the property is evaluated most closely and at smallest temperature increments at a temperature from just below to just above the temperature of transition. The need exists therefore for a device and a method of obtaining the optical property of the liquid in a way that accounts for all temperature data points continuously or at sufficient resolution to approximate a continuous measurement from a predetermined first temperature to a predetermined second temperature.
The preferred application of the invention is for liquids containing biological macromolecules. The need exists for a device and method allowing rapid characterization of the thermodynamics of biological macromolecule solutes and their interactions. Such characterizations are useful for drug design, design of probe molecules for use in high-throughput screening, protein engineering, and nucleic acid based diagnostics. Further applications of the method and device in proteomics, genomics and material science are anticipated.